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Alveolar bone regeneration for immediate implantplacement using an injectable bone substitute: an

experimental study in dogs.Damien Boix, Olivier Gauthier, Jérôme Guicheux, Paul Pilet, Pierre Weiss,

Gaël Grimandi, Guy Daculsi

To cite this version:Damien Boix, Olivier Gauthier, Jérôme Guicheux, Paul Pilet, Pierre Weiss, et al.. Alveolar boneregeneration for immediate implant placement using an injectable bone substitute: an experimentalstudy in dogs.. Journal of periodontology, American Academy of Periodontology, 2004, 75 (5), pp.663-71. �inserm-00176539�

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TITLE PAGE

Alveolar bone regeneration for immediate implant placement using an injectable bone

substitute: an experimental study in dog.

Damien Boix,* Olivier Gauthier,*† Paul Pilet,‡ Jérôme Guicheux,* Pierre Weiss,* Gaël

Grimandi,* Guy Daculsi*

* EM INSERM 99-03, Research Center on Materials with Biological Interest, Nantes, France.

† Surgery Department of the National Veterinary School, Nantes, France.

‡ Electron Microscopy Center, University Hospital, Nantes, France.

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ABSTRACT

Background

The aim of the present study was to assess the efficacy of a ready-to-use injectable bone

substitute for bone regeneration around dental implants placed into fresh extraction sockets.

Methods

Third and fourth mandibular premolars were extracted from 3 Beagle dogs and the

interradicular septa were surgically reduced to induce a mesial bone defect. Thereafter,

immediate placements of titanium implants were performed. On the left side of the jaw,

mesial bone defects were filled with an injectable bone substitute (IBS), obtained by

combining a polymer and a biphasic calcium phosphate ceramic. As a control, the right

defects were left unfilled. After 3 months of healing, specimens were prepared for histological

and histomorphometric evaluations.

Results

No post surgical complication was observed during the healing period. In all experimental

conditions, histological observations revealed a lamellar bone formation in contact with the

implant. Histomorphometric analysis showed that IBS triggers a significant (p<0.05) increase

in term of thread numbers in contact with bone (TN), bone-to-implant contact (BIC) and peri-

implant bone density (PBD), of about 8.6%, 11.0% and 14.7%, respectively. In addition, no

significant difference was observed when TN, BIC and PBD in filled defects were compared

to no-defect sites.

Conclusion

It is concluded that an injectable bone substitute composed of a polymeric carrier and calcium

phosphate significantly increase bone regeneration around immediate implants.

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KEY WORDS

Bone substitutes

Bone regeneration

Calcium phosphates

Dental implants

Osseointegration

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INTRODUCTION

Delayed placement of dental implants after alveolar healing is a proven and aesthetic

technique, but requires an extended time treatment before patients can enjoy the final result.1,2

Immediate implantation after tooth extraction is an attractive alternative that presents several

advantages such as reduction of post-extraction resorption, optimal positioning of the implant

and reduction of the time required for prosthetic rehabilitation.3,4 However, the discrepancy

between the transversal diameter of the socket (conical) and of the implant (cylindrical)

frequently generates a gap between the bony walls of the socket and the implant.5 In large

bony defects, this void can be colonised by epithelial cells, which induces fibrointegration and

then implant failure.6,7 Therefore, guided bone regeneration (GBR) alone or in association

with bone replacement graft has been developed and currently gives satisfactory results to

achieve implant osseointegration.7-9 Nevertheless, an early exposure of the barrier membrane

may promote secondary infections and consequently delay the peri-implant bone

regeneration.10,11 Rotated full thickness flaps were suggested as an alternative to GBR, but

vestibular flaps were often a source of aesthetical problems and palatal flaps may provoke

pain on exposed bone.12,13

Among the available materials used for pre-implant bone reconstruction, autologous bone is

currently the gold-standard because it is a source of osseous matrix, cells, and growth

modulating molecules.14 However, it requires the graft to be harvested at a distance from the

operation site, which makes the initial operation more complicated. To overcome the

autograft limits, many substitution biomaterials have been proposed. Materials of human and

animal origin have the disadvantages of limited supply and potential risk of cross

contamination.15,16 Consequently, synthetic products were developed.17 For example, biphasic

calcium phosphate (BCP), an intimate mixture of hydroxyapatite (HA) and ß-tricalcium

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phosphate (ß-TCP).18 BCP offers the potential for bone reconstruction since it has a chemical

composition close to biologic bone apatites19 and has already proven its efficacy as a bone

substitute material in many human clinical applications.20-24 More recently, the association of

BCP granules to a polymeric gel has provided a ready-to-use injectable bone substitute

material (IBS).25,26 Previous animal studies conducted in our laboratory have shown the

biocompatibility and the osteoconductive property of IBS.27-29 We have also demonstrated in

a preliminary study the IBS biofunctionality when used as socket filler in dogs.30

The aim of the present study was to assess the IBS influence on bone regeneration around

titanium dental implants immediately placed after tooth extraction without using an

osteopromotive membrane technique. A comparative histomorphometric study was performed

on peri-implant bone defects. Defects were treated by IBS filling and the control defects were

left unfilled to observe a physiological healing process.

MATERIALS AND METHODS

Injectable Bone Substi tute

The biomaterial used in this study was a composite combining a polymer and a calcium

phosphate mineral phase.31

Mineral phase

The ceramic composed of BCP granules with a 60/40 HA/ß-TCP weight ratio was obtained

by hydrolysis of a commercial dicalcium phosphate dihydrate. The resulting apatite powders

were granulated and sifted to select only granules with a 40 to 80 µm diameter. After sintering

at 1,150°C to form BCP, the granules were placed on a 40 µm sifter to eliminate the smallest

particles.

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Polymeric phase

As previously described,25 the associated polymer was a cellulose derivative (methyl-

hydroxy-propyl-cellulose = MHPC) previously proposed as an efficient vehicle for the

mineral phase.32 Briefly, a solution of 3% MPHC was prepared by dissolving raw dry MPHC

powder§ in double distilled water with stirring for 48 hours.

§ E4M PREMIUM EP, Colorcon, Bougival, France

Composite

The composite biomaterial, obtained by mixing a 3% MPHC solution with the 40 to 80 µm

BCP granules in a 50/50 weight ratio, was placed in ready-to-use plastic injectors and

sterilized by steam at 121°C for 20 minutes. This process preserves the gel consistency of the

polymer and decreases viscosity only very slightly.30

Animal experiments

The three 4-year-old female adult Beagle dogs used were bred exclusively for biomedical

studies and kept at the National Veterinary School of Nantes according to European

Community guidelines for the care and the use of laboratory animals (DE 86/609/CEE).

Gingival health was checked before experimentation, and teeth were scaled and polished

under general anaesthesia. Antibiotic treatment with spiramycin and metronidazole was

given for 5 days.

�Stomorgyl, Rhône-Mérieux, Lyon, France

Surgical procedures

All surgical procedures were performed under general anaesthesia with intravenous sodium

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thiopental¶ (12.5 mg/kg) followed by volatile anaesthesia with halothane. During surgery,

animals received 1 g of cephalosporin antibiotic# perfused intravenously. Implant placements

were performed on right and left third and fourth mandibular premolars (fig. 1-a).

Gingival incisions were performed mesially from the first premolar and distally to the molar

teeth. Then, full-thickness buccal and lingual flaps were raised. After vertical interradicular

section, each root was carefully elavated and then gently extracted.

Distal sockets were then drilled following the basic surgical principles governing the

placement of ITI dental implants.33 Thereafter, the interradicular septa were resected with a

mini-rongeur Friedman to create a mesial bone defect adjacent to the mesial socket on a 6 mm

height, 4 mm in the bucco-lingual direction and 5 mm in the mesio-distal direction (fig. 1-b).

After a complete alveolar cleaning, solid cylindrical screw implants** were manually inserted

in the implant beds (fig. 1-c). Specifications of ITI implants included a 3.3 mm diameter, an 8

mm length and a titanium plasma spray (TPS) coating. The primary stability was assessed and

implants were covered with 1.5 mm closure screws. On the left side, mesial sockets were

filled with IBS injection from the bottom to the top of the defect (fig. 1-c) in direct contact

with the implant (fig. 1-d). On the right side, implants were placed following the same

procedure but without IBS. The muccoperiosteal flaps were then sutured with an interrupted

non-absorbent suture†† (fig. 1-e).

Antibiotic treatment by intramuscular injection of cephalosporin (15 mg/kg, b.i.d.) was

continued for 48 hours after surgery. Animals were checked daily and fed with a soft diet.

Sutures were removed under short general anaesthesia 3 weeks later (day 21), and a normal

diet was then given. The animals were sacrificed 3 months after implantation (day 91) by

intravenous injection of overdosed sodium pentobarbital‡‡.

¶ Nesdonal, Rhône-Mérieux

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# Cefaloject, Bristol Laboratories, Paris, France

** ITI Esthetic plus, Straumann AG Institute, Waldenburg, Switzerland

†† Silk black braided, PRED, Paris, France

‡‡ Doléthal, Vétoquinol laboratory, Lure, France

Sample preparation

Bone segments were immediately dissected from animals with a diamond disk and fixed in a

4% paraformaldehyde solution buffered with phosphate buffered saline (PBS; pH 7.2). Each

sample was then dehydrated by successive immersion in graded ethanol for 24 hours at 4°C

(80°, 95°, 100°) and acetone (1 day). Thereafter, specimen were infiltrated with a

glycolmethylmetacrylate resin (GMMA) at -20°C for 8 days (repeated twice). Finally,

samples were embedded in GMMA (4 days, 4 °C).

Histological evaluation

Both filled and unfilled sites were histologically evaluated with light§§ and scanning

electron microscopy (SEM). For each sample, 10 µm thick sections were cut with a

diamond microtome saw¶¶ along to the long axis of implants in a mesiodistal direction and

then stained with Movat pentachrome for light microscope observation. Then, three 500 µm

sections were isolated from each sample and both surfaces were prepared by sputtering with

gold-palladium## for SEM observations at 20 kV.

§§ Axioplan 2, Zeiss, Jena, Germany

JSM 6300, Jeol, Tokyo, Japan

¶¶ MS 1600, Leitz, Westlar, Germany

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## AE1230, EMScope, Ashford, UK

Histomorphometric studies

For quantitative evaluation of bone regeneration, 500 µm sections prepared as described

above were observed with SEM apparatus coupled to a semi-automatic image analyser*** .

With image analysis, three parameters were measured in mesial bone defects and distal sites:

- The number of threads in contact with bone in relation to the total number of threads (TN),

- The Bone-to-Implant Contact (BIC) with a percentage ratio studied at two different heights:

. the endosseous part of the implant (EH)

. a 3 mm cervical height limited to the bone defects (DH),

- The Peri-implant-Bone-Density (PBD) inside DH on 3 zones at a 0.5 mm thickness:

. zone a : from the implant surface to 0.5 mm,

. zone b : from 0.51 to 1 mm,

. zone c : from 1.1 to 1.5 mm.

*** Quantimeter 500, Leica, Cambridge, UK

X-ray micro-analysis

Sample sections (500 µm thick) were polished in order to eliminate gold-palladium and were

sputter coated with carbon†††. Then, a quantitative X-ray micro-analysis was performed with

Energy Dispersive System (EDS)‡‡‡. Calcium (Ca), phosphate (P), magnesium (Mg), and

sodium (Na) values were measured at four different locations with regard to bone defects

(IBS filled and unfilled), alveolar bone (in implant proximity) and basal bone.

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††† JEE-4B, Jeol

‡‡‡ EXL II, Oxford instruments, London, UK

Statistical analysis

Results were expressed as mean (± SEM). Comparative study of means of the 16 filled sites,

the 18 unfilled sites and the 34 distal sites were performed using one way analysis of variance

(ANOVA) followed by a post-hoc test (Fischer’s projected least significant differences) with

a statistical significance at p<0.05.

RESULTS

Clinical results

No post surgical complication or infection was observed during the healing period. Sutures

were removed after three weeks. After one month, the closure screws initially covered by

muccoperiosteal flaps were exposed with excellent peri-implant soft tissue conditions (fig. 2).

At the time of euthanasia, all implants seemed to be clinically osseointegrated and none

presented with mobility.

Histological results

Light microscopy observations (magnification x 50) showed homogeneous bone healing in all

tested conditions (fig. 3). A bone-to-implant contact was observed on all the distal threads and

on most mesial threads in filled defects. An exposure of the first or the second thread was

observed in five unfilled defects. Higher magnification (x200) confirmed the close contacts

between lamellar bone and implant TPS surface (fig. 4). As previously described34, in the

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filled defects (fig. 4a), BCP granules were not visible and bone tissue showed the same

histological characteristics observed in the distal sites (fig. 4b). For all implants, junctional

epithelium was closely adapted to the machined smooth neck of the implant.

SEM showed new bone formation more homogenous and dense in the filled sites (fig. 5a) as

compare to the unfilled sites (fig. 5b). Large unmineralised areas were observed in absence of

IBS (fig. 6b), whereas bone tissue occupied a large extent of the filled sites (fig. 6a).

Quantitative evaluation

For all distal sites, threads were always in contact with bone surface. On the contrary, TN was

always lower in mesial bone defects. However, in mesial bone defects, TN remained always

significantly (p<0.01) higher in the presence of IBS as compare to the unfilled defect (fig. 7)

(98.44% ±1.52 and 92.78% ±2.98 respectively).

For EH, BIC in filled defects showed a significant (p<0.01) increase when compared with the

unfilled defects (54.54% ±1.29 and 46.67% ±1.08 respectively). In addition, a significant

augmentation of BIC in DH was observed in filled sites (48.86% ±1.97 vs 37.60% ±1.58 in

unfilled defects). Interestingly, BIC was not significantly different between the filled mesial

sites and the distal sites (fig. 8).

In all sites, PBD was always significantly lower for the zone close to the implant (zone a) as

compared to the zones located at a distance (zones b and c). No significant difference was

observed in zone b and zone c. Whatever the studied zone, minor PBD was obtained in

unfilled defects. IBS induced a significant (p<0.01) increase of PBD in zone a, b and c:

58.68% ±2.32 vs 44.5% ±1.76; 60.1% ±1.48 vs 48.46% ±1.51 and 61.71% ±0.85 vs 48.21%

±1.68 respectively. Furthermore, there was no significant difference between PBD values in

mesial filled sites and those of distal sites (fig. 9).

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The atomic composition of the analysed samples in EDS showed no significant difference

with respect to the studied locations. All the measurements in the implant peripheries were

very close to basal bone values. Calcium and phosphate values were respectively of 23.29%

±1.95 and 13.81% ±0.96 in filled bone defects. Magnesium and sodium represented less than

1% of the total elements. Ca/P ratio showed very little variation (from 1.66 ±0.03 for basal

bone to 1.70 ±0.07 in distal sites). In the filled and unfilled defects, this ratio was similar

(1.68 ±0.04 with IBS and 1.68 ±0.06 without IBS).

DISCUSSION

To our knowledge, this study demonstrates for the first time that the use of an injectable bone

substitute, composed of a calcium phosphate ceramic and a polymeric carrier, promotes bone

regeneration around dental implants immediately placed into fresh extractions sockets.

Cortical and trabecular bone grafts are the materials of choice for pre-implant surgery. Among

the biomaterials proposed as an alternative to bone autografts, calcium phosphate (CaP)

ceramics such as hydroxyapatite (HA), beta-tricalcium phosphate (ß-TCP) and the HA/ß-TCP

association [termed biphasic calcium phosphate (BCP)] have been used successfully because

their chemical composition is closely related to that of bone mineral.19 Within the past few

years, a proposed ready-to-use injectable bone substitute (IBS) has been developed. IBS is an

association of BCP granules with a cellulosic hydrogel.25 Our laboratory has previously

demonstrated the osteoconductive potential of this innovative biomaterial in an alveolar bone

site.30,35

In contrast to the delayed implantation protocol, immediate implant placement presents

numerous advantages including prevention of alveolar bone resorption and reduction of

rehabilitation time.3,4 Nevertheless, the success rate of both methods remains similar when

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primary stability of implants is associated with an osteopromotive technique.36,37 To favour

bone healing in cases where there are wide gaps between implant surfaces and sockets walls,

guided bone regeneration (GBR), alone or in association with bone substitute materials, has

been investigated with satisfactory but variable results.5,10 In an attempt to propose an

alternative to occlusive barrier membranes, the present work investigates the efficiency of IBS

to promote bone regeneration around solid-screw implants placed immediately after tooth

extraction.

Immediate implantation in dogs has displayed variable failure rates.10,38 The present study has

shown no implant mobility and no implant failure. This clinical success rate is comparable to

that obtained with delayed implants placed after a 3 month healing period.39 Several

hypotheses may explain these encouraging results: atraumatic extraction with respect to

alveolar walls; no bone heating during the defect creation thanks to the manual rongeur;

implant primary stability through preservation of vestibular and lingual processes;

postoperative soft diet to reduce mastication forces and daily postoperative observation to

prevent gingival inflammation.

To strengthen our clinical data, we conducted histological examinations of peri-implant

tissues regeneration. SEM and light microscopic observations showed mucosa, osteoid and

mineralised lamellar bone, but no residual BCP particles despite high magnification, as

previously reported.34 Furthermore, EDS demonstrated that newly-formed bone presents the

same Ca and P values as well as Ca/P ratio with regards to basal bone. Thus, IBS confirmed

his osteoconductive potential previously demonstrated.30,35 Nevertheless, the lack of

information during intermediary intervals did not permit evaluation of the biomaterial

transformation kinetics, which however seemed to be compatible with healing periods

currently recommended for immediate implantation.5,40

Numerous unmineralised areas were observed in unfilled defects witch suggest a delay in

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bone healing. However, partial bone regeneration was observed in these sites. In filled sites,

bone regeneration showed the same histological characteristics as compared to no-defect sites.

Interestingly, the absence of fibrous interposition in filled defects strongly suggest that IBS

may represent an occlusive barrier against epithelial cell colonisation.

The main objective of this work was to obtain reliable quantitative assessment of the different

parameters of bone regeneration: the number of osseointegrated threads (TN), the rate of

bone-to-implant contact (BIC), and the peri-implant bone density (PBD). Among the

numerous available tools, we focused on semi-automatic image analysis because it permits

quick and accurate analysis of a large amount of information.41,42 Previous

histomorphometrical studies, dedicated to implant osseointegration in animals, have mostly

been restricted to BIC evaluation.43,44 In order to further evaluate the bone regeneration

process beyond the bone/implant interface, image analysis was also conducted on PBD at

various distances from the implant surface.

A centripetal bone regeneration from the alveolar walls is even observed in unfilled sites

because experimental defects were not critical-size defects.30 However, all quantitative data

remains lower in unfilled defects. In addition, TN, BIC and PBD in filled defects

demonstrated no significant difference as compared to the no-defect sites. Since the

quantitative results in IBS-treated sites are higher or equal to the values reported with

immediate implantation in dog,10,44-47 IBS appears to satisfy immediate implantation

requirements.

CONCLUSION

This study demonstrates that our injectable bone substitute (IBS), a composite biomaterial,

promotes bone regeneration around implants immediately placed after tooth extraction.

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Therefore, it seems conceivable that IBS could be an alternative to autologous grafts or GBR

alone or in association with bone substitute biomaterial. However, these promising results are

to be confirmed by further biomechanical studies after implant loading, pull out tests and by

evaluation of reverse torque. IBS could be further propose for clinical applications such as

socket filling for ridge preservation, pre-implant reconstruction of osseous deficiencies or

sinus floor elevations.

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ACKNOWLEDGEMENTS

The authors gratefully acknowledge Dr J.M. Bouler for providing us with calcium phosphate

material and for his helpful contribution to EDS analysis.

The authors wish to thank the Straumann AG Institute (Waldenburg, CH) for providing ITI

dental implants.

The authors wish to thank Drs Julie Kazimiroff and Racquel Z LeGeros (Department of

Biomaterials and Biomimetics, New York University College of Dentistry) and Ms Karine

Sinander for their editorial assistance.

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37. Becker W, Becker BE, Ricci A, et al. A prospective multicenter clinical trial

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38. Levy D, Deporter DA, Pilliar RM, Watson PA, Valiquette N. Initial healing in the dog

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39. Cochran DL, Nummikoski PV, Higginbottom FL, Hermann JS, Makins SR, Buser D.

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40. Cosci F, Cosci B. A 7-year retrospective study of 423 immediate implants. Compend

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41. Gauthier O, Bouler JM, Aguado E, Pilet P, Daculsi G. Macroporous biphasic calcium

phosphate ceramics: influence of macropore diameter and macroporosity percentage

on bone ingrowth. Biomaterials 1998;19:133-139.

42. Nefussi JR, Ollivier A, Oboeuf M, Forest N. Rapid nodule evaluation computer-aided

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43. Barzilay I, Graser GN, Iranpour B, Natiella JR, Proskin HM. Immediate implantation

of pure titanium implants into extraction sockets of Macaca fascicularis. Part II:

Histologic observations. Int J Oral Maxillofac Implants 1996;11:489-497.

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between bone and immediate endosseous implants: a pilot study in dogs. Int J Oral

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hydroxyapatite-coated and titanium-coated implants in rabbit bone. Int J Oral

Maxillofac Implants 1992;7:485-490.

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generation around implants placed into fresh extraction sockets: an experimental study

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histomorphometric comparison of immediately placed hydroxyapatite-coated and

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REPRINT ADRESS

Send reprint requests to: Dr. Pierre Weiss, EM INSERM 99-03, Centre de Recherche sur les

Matériaux d’Intérêt Biologique, 1 place Alexis Ricordeau, 44042 Nantes cedex 1, France.

Fax: 33-2-40-08-37-12; e-mail: pweiss@sante.univ-nantes.fr

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ILLUSTRATION LEGENDS

Figure 1: Photographs of the surgical procedures.

a: third (P3) and fourth (P4) left mandibular premolar (preoperative view),

b: mesial socket after extraction (MS), interadicular septum resection (SR), distal socket

preparation (DS) in P3 and mesial bone defect (MS + SR) probing in P4,

c: implants (ITI) placed in distal socket of P3 before filling with IBS and IBS injection in

mesial bone defect of P4,

d: 2 implants placed in distal sockets and non-overpacked fill with IBS,

e: overlapping flap sutures.

Figure 2: Photograph one month after surgery. Note the soft tissues healing around closure

screws emergence.

Figure 3: Light microscopy observation of an axial section of implant after three months of

healing with IBS. Note the homogeneous bone healing around implant.

(Movat pentachrome, original magnification x 50).

Figure 4: Light microscopy observations of the first cervical thread of implants after three

months of healing. Note different bone regenerations at the implant contact.

a: mesial defect filled by IBS,

b: distal site without defect,

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c: mesial defect unfilled by IBS.

(Movat pentachrome, original magnification x 200).

Figure 5: Scanning electron microscopy observations of axial sections of implants after three

months of healing.

a: mesial filled defect (left) and distal site (right). Note the presence of an homogeneous bone

ingrowth in the filled defect when compared with distal site,

b: unfilled mesial defect (left) and distal site (right). Note the presence of decreased bone

regeneration in mesial unfilled defect when compared with distal site.

(bar = 1 mm).

Figure 6: Scanning electron microscopy observations of the cervical threads of implants after

three months of healing.

a: filled defect. Note the close contact between bone and TPS surface

b: unfilled defect. Note the presence of large unmineralised areas

(bar = 0.2 mm).

Figure 7: Threads of implants in contact with bone after three months of defect healing either

in sites filled (defects + IBS) or unfilled (unfilled defects) with IBS. As a control, distal sites

were left without bone defects (no-defect). Results are expressed in percentage (± SEM) of

threads not in contact with bone.

* : p < 0.01 compared with the unfilled defects.

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Figure 8: Bone-to-implant contact in endo-osseous height of implants (EH) and in bone defect

height (DH) after three months of defect healing either in sites filled (filled defects) or

unfilled (unfilled defects) with IBS. As a control, distal sites were left without bone defects

(no-defect). Results are expressed in height percentage (± SEM).

*: p < 0.01 compared with the unfilled defects.

NS: no significant

Fig. 9: Peri-implant bone density of 3 various zones after three months of defect healing either

in sites filled (filled defects) or unfilled (unfilled defects) with IBS. As a control, distal sites

were left without bone defects (no-defect). As described in materials and methods, zone a was

from TPS surface to 0.5 mm, zone b, from 0.51 to 1 mm, zone c, from 1.1 to 1.5 mm. Results

are expressed as percentage (± SEM) of total surface in each zone.

* : p < 0.01 compared with the unfilled defects.

NS: no significant

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